Human immunodeficiency virus type-1 (HIV-1) is a retrovirus that leads to the development of acquired immune deficiency syndrome (AIDS). The infectivity rate of HIV in the United States has been estimated at about 40,000 new infections per year. Current treatments for HIV infection are designed to interfere with the ability of the virus to replicate by inhibiting either HIV protease or HIV reverse transcriptase (RT).
Efavirenz (SUSTIVA, Bristol-Meyers Squibb) is one of the FDA-approved drugs used in the treatment of patients infected with HIV. Efavirenz has been shown to lower the amount of HIV in the blood (the “viral load”). When taken with other anti-HIV medicines, efavirenz has been shown to reduce patients' viral load and to increase their CD4 cell count.
Clinical research has demonstrated that HIV can develop resistance to drugs used in HIV therapy, including efavirenz. Such drug resistance is thought to be a primary reason for therapy failure. The development of drug resistance in HIV may be a result of the virus rapid replication rate. Despite its potency, efavirenz has a low genetic barrier. A high level of phenotypic resistance can be induced by a single mutation, frequently at lysine 103 (K103N) in the RT gene. The emergence of efavirenz-resistant HIV mutants could be a result of repeated exposure to ineffective or sub-therapeutic drug levels.
Therapeutic failures are observed more frequently in patients having low serum concentrations of efavirenz. For example, Marzolini et al., AIDS 15 (London), 71-75, 2001, reported virological failure in 50% of patients (85 total patients) that had low plasma levels of efavirenz, e.g., <1000 μg/L. In patients with plasma levels of efavirenz that ranged from 1000-4000 μg/L, or more than 4000 μg/L, they observed virological failure in 18-22% of those patients. Moreover, 20-40% of patients receiving efavirenz reported central nervous system (CNS) side effects that include dizziness, hallucinations, nightmares, and insomnia. While these symptoms are usually mild to moderate in severity and are reported to subside progressively over a few weeks after the initiation of efavirenz therapy, it has been reported that about 4% of patients discontinue therapy because of the severity or persistence of these side effects. CNS toxicity was approximately three times more frequent in patients with high efavirenz levels, e.g., >4000 μg/L, compared with the patients having levels in the 1000-4000 μg/L range. This implies that treatment failure and CNS side effects are associated with low and high efavirenz plasma levels, respectively. The variability of efavirenz levels in individuals strongly supports that the dose adjustment should be based on therapeutic drug monitoring (TDM) in order to optimize beneficial therapeutic effects while minimizing CNS side effects.
As pharmacological differences among patients introduce wide heterogeneity in the response to antiviral therapy, monitoring of drug levels could be useful in the management of HIV infection as well as the disorders and diseases associated with HIV infection. Formal therapeutic drug monitoring of antiviral drugs useful in HIV therapy is known using high-performance liquid chromatographic (HPLC) methods (Marzolini et al., ibid.).
While HPLC methods can be used to determine efavirenz levels in plasma, such methods are impractical for commercial use due to, for example, long sample preparation time, long assay time, high cost, and labor-intensive procedures. Thus, a simple and fast analytical method for measurement of plasma levels of efavirenz is needed for effective TDM. Immunoassay techniques are well suited for such analytical applications.
Standard medical practice for treatment of HIV infections is to co-administer three HIV drugs, at least two of which come from different classes, for example, 2 nucleoside reverse transcriptase inhibitors (NRTI's) and a non-nucleoside reverse transcriptase inhibitor (NNRTI). Thus, for an immunoassay technique for measuring efavirenz, it is essential that an antibody be highly specific for efavirenz.